1. Field of Invention
This invention pertains to a switchable photosystem-II (PSII) designer organism that is specifically designed for enhanced photobiological production of molecular hydrogen (H2). The various embodiments include (1) application of a genetic switch that can control the expression of PSII activity for production of H2 without O2 production and (2) combination of switchable PSII with programmable proton channels in algal photosynthetic membrane that further enhance photobiological H2 production. In particular, this invention pertains to a switchable PSII designer alga for enhanced photobiological H2 production. Various embodiments solves the following six major problems that currently challenge those in the field of photobiological H2 production: (1) drainage of electrons by O2, (2) poisoning of the hydrogenase enzyme by O2, (3) the mixed H2 and O2 gas-product separation and safety issues, (4) restriction of photosynthetic H2 production by accumulation of a proton gradient, (5) competitive inhibition of photosynthetic H2 production by CO2, and (6) requirement for bicarbonate binding at photosystem II (PSII) for efficient photosynthetic activity.
2. Background
Algal (such as Chlamydomonas reinhardtii, Platymonas subcordiformis, Chlorella fusca, Ankistrodesmus braunii, and Scenedesmus obliquus) photosynthetic hydrogen (H2) production from water has tremendous potential to be a clean and renewable energy resource. As shown in FIG. 1, in the algal system, H2 is produced through hydrogenase-catalyzed reduction of protons by the electrons generated from photosynthetic oxidation of water using sunlight energy. The net result is photoevolution of H2 and O2 from water (H2O). However, there are a number of technical issues that must be addressed before algal H2 production can become practical. The following lists the six technical problems that currently challenge researchers and investors in the field of photosynthetic H2 production: (1) drainage of electrons by O2, (2) poisoning of the hydrogenase enzyme by O2, (3) the mixed H2 and O2 gas-product separation and safety issues, (4) restriction of photosynthetic H2 production by accumulation of a proton gradient, (5) competitive inhibition of photosynthetic H2 production by CO2, and (6) requirement for bicarbonate binding at photosystem II (PSII) for efficient photosynthetic activity.
Of these six technical problems in the wild-type organism, the first three are all oxygen-related issues that must be solved in order for the algal H2-production technology to work. Here is the reason why the three oxygen-related issues must be solved in order for the algal H2-production technology to work well. In wild-type algae as illustrated in FIG. 1, photosynthetic water splitting under anaerobic condition can result in simultaneous production of H2 and O2 in the same cell. Therefore, the gas products in this case are a dangerous mixture of H2 and O2 that requires safe separation (problem 3). Furthermore, the O2 produced in the alga can inhibit H2 production by two mechanisms: acting as an electron acceptor [possibly through the RuBisco (which is also a known oxygenase) enzyme at the Calvin cycle] that drains the electrons away from the Fd/hydrogenase H2 production pathway (problem 1), and poisoning the hydrogenase enzyme directly (problem 2).
The inhibition of algal H2 production by O2 is a serious problem that can be measured experimentally. As shown in the experimental data presented in FIG. 2, an introduction of merely 0.1000% O2 (equivalent to 1000 ppm O2) results in a dramatic inhibition on the H2-production rate of the wild-type alga C. reinhardtii. The steady-state H2-production rate in the presence of 1000 ppm O2 was 0.33 μmol of H2/mg Chl/hr, which is only about 2.8% (less than a 30th) of the full steady-state rate (12 μmol of H2/mg Chl/hr) before the introduction of the 0.1000% O2. Therefore, it is not practical to use the wild-type alga for production of H2 and O2 by photosynthetic water splitting. In order to realize the full potential of the algal H2-production mechanism, these oxygen-related problems must be solved.
The various embodiments overcome these three oxygen-related problems for enhanced H2 production in an effective way by creating a designer alga that contains switchable PSII reaction centers for production of pure H2 without O2 production. Use of the designer switchable PSII algae in conjunction with the features of my designer proton-channel algae represents a significant photobiological H2-production technology in the field of renewable energy production. The application titled “Designer proton-channel transgenic algae for photobiological hydrogen production” and filed on the same day and by the same inventor as this application is hereby incorporated by reference in its entirety. There are other approaches reported in the field of studies such as the “sulfur-deprivation”-based approach that are also attempted to solve the O2-related problems. The various embodiments are distinguished from the prior art by its innovative application of the emerging RNA interference technique with a genetic switch to selectively (and switchably) suppress PSII oxygen-evolution activity and create free proton channels in algal photosynthetic membrane. It provides a unique solution to both the oxygen-related problems and the proton-gradient-related issues for enhanced photobiological H2 production. For example, the “sulfur-deprivation”-based physiological approach is not required here at all. With its features of switchable genetic expression of both PSII and proton channel in algal photosynthetic membrane, the designer alga can be used to solve all of the listed six problems that currently challenge researchers and investors in the field of photobiological H2 production.